海洋生态与环境科学重点实验室知识库

链状亚历山大藻（Alexandrium catenella）和太平洋亚历山大藻（Alexandrium pacificum）分别是我国渤海和黄海、东海和南海亚历山大藻藻华的主要肇事种。近年来，我国近海这两种亚历山大藻频发藻华，其产生的麻痹性贝毒（Paralytic shellfish toxins，PST）常造成贝类染毒，不时造成沿海居民因食用染毒的贝类而中毒，甚至死亡。亚历山大藻属内产毒和不产毒藻种形态学相似，传统形态学方法难于有效区分，而且藻华暴发时藻细胞丰度低，危害效应严重，迫切需要研发快速、简便、特异性的分子检测方法用于我国近海这两种亚历山大藻藻华现场检测和监测。本研究采用重组酶聚合酶扩增技术（Recombinase polymerase amplification，RPA），结合核酸快速提取方法和横向流动试纸条（Lateral flow dipstick，LFD），研发这两种亚历山大藻的定量快检方法。主要的研究结果如下：

核酸快速提取方法：采用重组酶聚合酶扩增技术（RPA）和聚合酶链式反应（Polymerase Chain Reaction）作为检测方法，对比分析了四种核酸快速提取法提取藻华微藻核酸的效果，发现DMSO-Triton X100快速提取法最优。该方法具有对RPA和PCR分子反应无抑制、操作简便、裂解条件温和、核酸提取效率高等优点，还具有一定的抗干扰能力，能从自然海水环境中提取藻华微藻的核酸，用于RPA和PCR等分子反应。

RPA-LFD定量快检方法：通过对亚历山大藻属的核糖体ITS区序列对比分析，分别设计并筛选链状亚历山大藻和太平洋亚历山大藻的特异性RPA引物对和探针，结合开发的核酸快提法和LFD灰度比值法，建立了这两种亚历山大藻的RPA-LFD定量快检方法。建立的定量快检方法可特异性检测目标藻种；灵敏度高，检出限都约为6 cells/L；检测范围广，跨4个数量级；具有不错的重复性和重现性；操作简单，不依赖复杂仪器；检测效率高，在37℃恒温条件下，90分钟以内完成对亚历山大藻DNA提取和丰度定量检测；通过对野外模拟样品回收率的检测，表明该方法有一定的抗干扰能力，具有野外应用潜力。

综上所述，本研究针对我国近海两种典型亚历山大藻——链状亚历山大藻和太平洋亚历山大藻建立了快速、简便、具有现场应用潜力的RPA-LFD定量快检方法，为这两种亚历山大藻藻华现场快速检测和监测提供重要的技术手段。

Alexandrium catenella and Alexandrium pacificum are the two predominant dinoflagellates responsible for paralytic shellfish toxins (PSTs) in ocean. In recent years, these two Alexandrium species have frequently led to harmful algal blooms (HABs) in China’s coastal waters, resulting in the contamination of shellfish with PSTs. Consumption of PST-contaminated shellfish by coastal residents poses a significant risk of poisoning and potential fatality. Due to the low cell abundance of Alexandrium during bloom events, and morphological similarities between toxic and non-toxic Alexandrium species, there is an urgent need to develop a rapid, simple and field-applicable molecular detection method with high specificity to detect and monitor blooms of these two toxic Alexandrium species in China’s coastal waters. This study presents the development and preliminary application of rapid quantitative detection methods for two Alexandrium species, utilizing Recombinase Polymerase Amplification (RPA) combined with rapid nucleic acid extraction methods and Lateral Flow Dipstick (LFD). The results of this study are summarized as follows:

Rapid nucleic acid extraction method: Two molecular biology techniques, RPA and polymerase chain reaction (PCR), were employed to analyze the effects of four rapid nucleic acid extraction methods on HAB species. The results indicated that the DMSO-Triton X100 rapid method developed in this study was the best one. The method presents several advantages, including non-interference with molecular reactions, straightforward operation, gentle lysis conditions, and high efficiency in nucleic acid extraction. Furthermore, it demonstrates a certain degree of resistance to interference, remains unaffected by environmental contaminants, and can consistently extract nucleic acids under natural seawater conditions.

RPA-LFD rapid quantitative methods: Ribosomal ITS region of the genus Alexandrium was subjected to comparative sequence analysis, followed by the design of RPA primer pairs and probes for A. catenella and A. pacificum, respectively; subsequently, the optimal combination of RPA primer pairs and probes were screened from the designed ones. RPA-LFD rapid quantitative methods for two Alexandrium species were developed using a combination with DMSO-Triton X100 nucleic rapid rapid extraction method, RPA and LFD grey scale ratio method. The developed RPA-LFD rapid quantitative methods for two Alexandrium species were capable of specifically amplifying the target Alexandrium species. They demonstrate high sensitivity, with detection limits of approximately 6 cells/L, and a wide detection range spanning four orders of magnitude. Furthermore, they exhibit stable reproducibility and repeatability, as well as a certain degree of anti-interference ability. In summary, the user-friendly RPA-LFD methods developed in this study do not rely on complex instrumentations, have high detection efficiency for DNA extraction and quantitative detection the cell abundance of these two Alexandrium species in just less than 90 minutes at 37℃. The impressive recovery rate observed in simulated field samples suggests that the developed RPA-LFD rapid quantitative methods exhibit a notable resistance to interference from environmental contaminants, indicating their potential for practical application in field.

In conclusion, this study has successfully established the two field-applicable RPA-LFD methods for the rapid quantitative detection of A. catenella and A. pacificum, predominant causative species of Alexandrium blooms in the coastal waters of China. These methods will serve as crucial tools for rapid on-site detection and monitoring of HAB incidents caused by these two Alexandrium species.

目  录

第1章 绪论... 1

1.1 亚历山大藻藻华概述... 1

1.1.1 亚历山大藻藻华发生情况及其危害... 1

1.1.2 链状亚历山大藻和太平洋亚历山大藻的分类修订... 2

1.2 亚历山大藻的检测技术研究现状... 2

1.2.1 基于形态结构的检测技术... 3

1.2.2 基于细胞色素的检测技术... 3

1.2.3 免疫分析技术... 3

1.2.4 核酸检测技术... 4

1.3 重组酶聚合酶扩增技术的发展及应用... 11

1.3.1 重组酶聚合酶扩增技术的发展... 11

1.3.2 重组酶聚合酶扩增产物的检测... 11

1.3.3 重组酶聚合酶扩增技术在藻华藻种检测中的应用... 13

1.4 核酸快速提取方法... 14

1.5 研究目的及意义... 15

第2章 核酸快速提取方法的建立... 17

2.1 前言... 17

2.2 材料与方法... 17

2.2.1 实验藻种及培养条件... 17

2.2.2 引物设计与合成... 18

2.2.3 核酸提取液筛选与配制... 18

2.2.4 微藻样品采集... 19

2.2.5 核酸提取... 19

2.2.6 RPA和PCR反应体系和产物分析... 20

2.2.7 不同核酸快提液对扩增反应影响的对比分析... 21

2.2.8 不同核酸快提液提取的DNA模板扩增反应效果的对比分析... 21

2.2.9 不同裂解温度和时长的核酸提取效果分析... 21

2.2.10 六种藻华微藻的核酸提取效果分析... 21

2.2.11 模拟野外样品核酸的提取效果分析... 21

2.3 结果... 22

2.3.1 不同核酸快提液对扩增反应影响的对比... 22

2.3.2 核酸快提液提取DNA模板对扩增反应的影响... 23

2.3.3 DMSO-Triton X100核酸快提液在不同裂解温度和时长下的核酸提取效果... 25

2.3.4 六种藻华微藻核酸的提取效果... 27

2.3.5 模拟野外样品核酸的提取效果... 27

2.4 讨论... 29

2.4.1 DMSO-Triton X100核酸快提法的特点... 29

2.4.2 DMSO-Triton X100核酸快提法的应用前景... 31

2.5 小结... 32

第3章 链状亚历山大藻RPA-LFD快速检测方法的建立... 33

3.1 前言... 33

3.2 材料与方法... 33

3.2.1 实验藻株及培养条件... 33

3.2.2 样品制备与核酸提取... 34

3.2.3 特异性引物、探针设计... 35

3.2.4 RPA反应体系建立和引物筛选... 36

3.2.5 RPA反应体系优化... 36

3.2.6 RPA方法特异性验证... 37

3.2.7 基于检测线与质控线灰度比值的定量检测有效性分析... 37

3.2.8 RPA-LFD方法标准曲线和工作曲线的建立... 37

3.2.9 RPA-LFD方法检出限的确定... 38

3.2.10 RPA-LFD方法重复性和重现性验证... 38

3.2.11 RPA-LFD方法模拟野外样品测试... 38

3.3 结果... 38

3.3.1 特异性引物、探针设计... 38

3.3.2 引物筛选... 39

3.3.3 RPA反应体系优化... 40

3.3.4 RPA方法特异性验证... 40

3.3.5 基于检测线与质控线灰度比值的定量检测有效性的分析... 43

3.3.6 RPA-LFD方法标准曲线和工作曲线的建立... 46

3.3.7 RPA-LFD方法检出限的确定... 47

3.3.8 RPA-LFD方法的重复性和重现性验证... 48

3.3.9 RPA-LFD方法模拟野外样品回收率的测试... 48

3.4 讨论... 49

3.5 小结... 51

第4章 太平洋亚历山大藻RPA-LFD快速检测方法的建立... 53

4.1 前言... 53

4.2 材料与方法... 53

4.2.1 实验藻株及培养条件... 53

4.2.2 样品制备与核酸提取... 54

4.2.3 特异性引物、探针设计... 54

4.2.4 RPA反应体系建立和引物筛选... 55

4.2.5 RPA反应体系优化... 55

4.2.6 RPA方法特异性验证... 55

4.2.7 RPA-LFD方法标准曲线和工作曲线建立... 55

4.2.8 RPA-LFD方法检出限确定... 55

4.2.9 RPA-LFD方法的重复性和重现性... 56

4.2.10 RPA-LFD方法模拟野外样品回收率的检测... 56

4.3 结果... 56

4.3.1 特异性引物、探针设计... 56

4.3.2 引物筛选... 57

4.3.3 RPA反应体系优化... 58

4.3.4 RPA方法特异性验证... 58

4.3.5 RPA-LFD方法标准曲线和工作曲线的建立... 61

4.3.6 RPA-LFD方法检出限的确定... 61

4.3.7 RPA-LFD方法的重复性和重现性验证... 62

4.3.8 RPA-LFD方法模拟野外样品回收率的测试... 63

4.4 讨论... 63

4.5 小结... 64

第5章 结论与展望... 65

5.1 结论.... 65

5.2 不足与展望... 65

参考文献... 67

附录 用于引物和探针设计的亚历山大藻属ITS区序列信息... 77

致谢... 79

作者简历及攻读学位期间发表的学术论文与其他相关学术成果... 81